The Effect of Ceramide on Model Membranes and Apoptotic Cells Determined by X-Ray Scattering, Solid State NMR, and Flow Cytometry

Abstract

Ceramides (Cer) are sphingolipids involved in the development of lung alveolar cell apoptosis (programmed death) and possibly in the clearance of apoptotic cells by alveolar macrophages. Typically, the clearance process is initiated by the binding of the phosphatidylserine (PS) receptor on the macrophage plasma membrane to PS which is externalized on the plasma membrane of the apoptotic (target) cell. We use a combination of molecular and cellular methods to determine the effect of ceramides on the ability of alveolar macrophages to engulf apoptotic cells. Engulfment experiments of labeled apoptotic Jurkat cells were performed with rat alveolar macrophages (AM) obtained via bronchoalveolar lavage. AM were treated with various ceramide species and efferocytosis was quantified by flow cytometry. Using small-angle X-ray scattering and solid state 2H NMR we determined how ceramides (C6:0, C18:1) affect the molecular organization and the physical properties of PS-containing membranes. By investigating model membranes with various Cer:PS:PC ratios and deuterated species we show how ceramides alter membrane thickness, bending rigidity, and the ordering of the lipid acyl chains. These studies can lead to a better understanding of the molecular mechanisms responsible for apoptotic cell clearance. If the clearance process is impaired, apoptotic cells may progress to secondary necrosis, resulting in release of harmful cellular contents and tissue inflammation. (IUPUI Membrane Biosciences Signature Center grant.)